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Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

Zhong Y, Brieher WM, Gumbiner BM - J. Cell Biol. (1999)

Bottom Line: Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.It does not work when added to CEC1-5 on the substrate.Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

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Analysis of mAb AA5 activity on cell lines and in vitro.  (A) Effect of mAb AA5 on C-cadherin–mediated adhesion of  CHO cells. C-CHO cells (expressing wild-type C-cadherin) were  harvested in the presence of calcium and then allowed to attach  to CEC1-5–coated capillary tube in the presence of either mAb  AA5 Fab or nonimmune mouse Fab for 30 min. Adhesive  strength was measured as the resistance of cell detachment to  progressively increasing flow rates. The experiment was performed in triplicate and the percentage of cells remaining ± SE  was plotted as a function of flow rate. (B) Effect of mAb AA5 on  the adhesive function of a cytoplasmic tail truncated C-cadherin  expressed in CHO cells (CT-CHO). Adhesion of CT-CHO cells  was assayed in the presence of either mAb AA5 Fab or nonimmune mouse Fab using the flow assay described in A. The effect of  inhibitory mAb 6B6 is shown for comparison. The experiment was  performed in triplicate and the percentage of cells remaining ±  SE was plotted as a function of time. (C) Effect of mAb AA5 on  the aggregation of CEC1-5–coated FluoSpheres. Dispersed  CEC1-5–coated FluoSpheres were incubated either with mAb  AA5 Fab or with nonimmune mouse IgG Fab in the presence of  calcium for various time periods. As a negative control, samples  were also incubated with the presence of EDTA. The number of  aggregated FluoSpheres (superthreshold particles) were counted  using a Coulter counter. The experiment was performed in triplicate and the number of superthreshold particles ± SE was plotted as a function of time.
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Figure 4: Analysis of mAb AA5 activity on cell lines and in vitro. (A) Effect of mAb AA5 on C-cadherin–mediated adhesion of CHO cells. C-CHO cells (expressing wild-type C-cadherin) were harvested in the presence of calcium and then allowed to attach to CEC1-5–coated capillary tube in the presence of either mAb AA5 Fab or nonimmune mouse Fab for 30 min. Adhesive strength was measured as the resistance of cell detachment to progressively increasing flow rates. The experiment was performed in triplicate and the percentage of cells remaining ± SE was plotted as a function of flow rate. (B) Effect of mAb AA5 on the adhesive function of a cytoplasmic tail truncated C-cadherin expressed in CHO cells (CT-CHO). Adhesion of CT-CHO cells was assayed in the presence of either mAb AA5 Fab or nonimmune mouse Fab using the flow assay described in A. The effect of inhibitory mAb 6B6 is shown for comparison. The experiment was performed in triplicate and the percentage of cells remaining ± SE was plotted as a function of time. (C) Effect of mAb AA5 on the aggregation of CEC1-5–coated FluoSpheres. Dispersed CEC1-5–coated FluoSpheres were incubated either with mAb AA5 Fab or with nonimmune mouse IgG Fab in the presence of calcium for various time periods. As a negative control, samples were also incubated with the presence of EDTA. The number of aggregated FluoSpheres (superthreshold particles) were counted using a Coulter counter. The experiment was performed in triplicate and the number of superthreshold particles ± SE was plotted as a function of time.

Mentions: Experiments were undertaken to determine how mAb AA5 activates C-cadherin. For integrins, there are two general classes of activating mAbs. One class, exemplified by anti-β3 mAbs including P41 (O'Toole et al., 1990), Ab 62 (O'Toole et al., 1990), and D3GP3 (Kouns et al., 1990), binds to and stabilizes a high-affinity conformation of the isolated integrin protein. The second class is more complex and requires cellular processes to activate the integrin (Shattil et al., 1985; Dransfield and Hogg, 1990). To determine whether mAb AA5 influences the homophilic binding function of the purified CEC1-5 ectodomain, its effect on the aggregation of CEC1-5–coated beads was assayed. Previous experiments showed that bead aggregation depended on calcium and the dimerization of CEC1-5 and was strongly inhibited by inhibitory anti–C-cadherin mAbs 6B6 and 5G5 (Brieher et al., 1996). In contrast, mAb AA5 weakly, but reproducibly enhanced the rate of CEC1-5–coated bead aggregation (Fig. 4 C). This suggests that mAb AA5 can affect to some extent the intrinsic homophilic binding function of the CEC1-5 ectodomain independent of cellular context.


Analysis of C-cadherin regulation during tissue morphogenesis with an activating antibody.

Zhong Y, Brieher WM, Gumbiner BM - J. Cell Biol. (1999)

Analysis of mAb AA5 activity on cell lines and in vitro.  (A) Effect of mAb AA5 on C-cadherin–mediated adhesion of  CHO cells. C-CHO cells (expressing wild-type C-cadherin) were  harvested in the presence of calcium and then allowed to attach  to CEC1-5–coated capillary tube in the presence of either mAb  AA5 Fab or nonimmune mouse Fab for 30 min. Adhesive  strength was measured as the resistance of cell detachment to  progressively increasing flow rates. The experiment was performed in triplicate and the percentage of cells remaining ± SE  was plotted as a function of flow rate. (B) Effect of mAb AA5 on  the adhesive function of a cytoplasmic tail truncated C-cadherin  expressed in CHO cells (CT-CHO). Adhesion of CT-CHO cells  was assayed in the presence of either mAb AA5 Fab or nonimmune mouse Fab using the flow assay described in A. The effect of  inhibitory mAb 6B6 is shown for comparison. The experiment was  performed in triplicate and the percentage of cells remaining ±  SE was plotted as a function of time. (C) Effect of mAb AA5 on  the aggregation of CEC1-5–coated FluoSpheres. Dispersed  CEC1-5–coated FluoSpheres were incubated either with mAb  AA5 Fab or with nonimmune mouse IgG Fab in the presence of  calcium for various time periods. As a negative control, samples  were also incubated with the presence of EDTA. The number of  aggregated FluoSpheres (superthreshold particles) were counted  using a Coulter counter. The experiment was performed in triplicate and the number of superthreshold particles ± SE was plotted as a function of time.
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Figure 4: Analysis of mAb AA5 activity on cell lines and in vitro. (A) Effect of mAb AA5 on C-cadherin–mediated adhesion of CHO cells. C-CHO cells (expressing wild-type C-cadherin) were harvested in the presence of calcium and then allowed to attach to CEC1-5–coated capillary tube in the presence of either mAb AA5 Fab or nonimmune mouse Fab for 30 min. Adhesive strength was measured as the resistance of cell detachment to progressively increasing flow rates. The experiment was performed in triplicate and the percentage of cells remaining ± SE was plotted as a function of flow rate. (B) Effect of mAb AA5 on the adhesive function of a cytoplasmic tail truncated C-cadherin expressed in CHO cells (CT-CHO). Adhesion of CT-CHO cells was assayed in the presence of either mAb AA5 Fab or nonimmune mouse Fab using the flow assay described in A. The effect of inhibitory mAb 6B6 is shown for comparison. The experiment was performed in triplicate and the percentage of cells remaining ± SE was plotted as a function of time. (C) Effect of mAb AA5 on the aggregation of CEC1-5–coated FluoSpheres. Dispersed CEC1-5–coated FluoSpheres were incubated either with mAb AA5 Fab or with nonimmune mouse IgG Fab in the presence of calcium for various time periods. As a negative control, samples were also incubated with the presence of EDTA. The number of aggregated FluoSpheres (superthreshold particles) were counted using a Coulter counter. The experiment was performed in triplicate and the number of superthreshold particles ± SE was plotted as a function of time.
Mentions: Experiments were undertaken to determine how mAb AA5 activates C-cadherin. For integrins, there are two general classes of activating mAbs. One class, exemplified by anti-β3 mAbs including P41 (O'Toole et al., 1990), Ab 62 (O'Toole et al., 1990), and D3GP3 (Kouns et al., 1990), binds to and stabilizes a high-affinity conformation of the isolated integrin protein. The second class is more complex and requires cellular processes to activate the integrin (Shattil et al., 1985; Dransfield and Hogg, 1990). To determine whether mAb AA5 influences the homophilic binding function of the purified CEC1-5 ectodomain, its effect on the aggregation of CEC1-5–coated beads was assayed. Previous experiments showed that bead aggregation depended on calcium and the dimerization of CEC1-5 and was strongly inhibited by inhibitory anti–C-cadherin mAbs 6B6 and 5G5 (Brieher et al., 1996). In contrast, mAb AA5 weakly, but reproducibly enhanced the rate of CEC1-5–coated bead aggregation (Fig. 4 C). This suggests that mAb AA5 can affect to some extent the intrinsic homophilic binding function of the CEC1-5 ectodomain independent of cellular context.

Bottom Line: Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation.It does not work when added to CEC1-5 on the substrate.Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York 10021, USA.

ABSTRACT
The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate the role of cadherin regulation in morphogenesis and to begin to analyze the molecular mechanisms of cadherin regulation, we have screened for monoclonal antibodies (mAbs) that allow us to manipulate the adhesive state of the cadherin molecule. Xenopus C-cadherin is regulated during convergent extension movements of gastrulation. Treatment of animal pole tissue explants (animal caps) with the mesoderm-inducing factor activin induces tissue elongation and decreases the strength of C-cadherin-mediated adhesion between blastomeres (Brieher, W.M., and B.M. Gumbiner. 1994. J. Cell Biol. 126:519-527). We have generated a mAb to C-cadherin, AA5, that restores strong adhesion to activin-treated blastomeres. This C-cadherin activating antibody strongly inhibits the elongation of animal caps in response to activin without affecting mesodermal gene expression. Thus, the activin-induced decrease in C-cadherin adhesive activity appears to be required for animal cap elongation. Regulation of C-cadherin and its activation by mAb AA5 involve changes in the state of C-cadherin that encompass more than changes in its homophilic binding site. Although mAb AA5 elicited a small enhancement in the functional activity of the soluble C-cadherin ectodomain (CEC1-5), it was not able to restore cell adhesion activity to mutant C-cadherin lacking its cytoplasmic tail. Furthermore, activin treatment regulates the adhesion of Xenopus blastomeres to surfaces coated with two other anti-C-cadherin mAbs, even though these antibodies probably do not mediate adhesion through a normal homophilic binding mechanism. Moreover, mAb AA5 restores strong adhesion to these antibodies. mAb AA5 only activates adhesion of blastomeres to immobilized CEC1-5 when it binds to C-cadherin on the cell surface. It does not work when added to CEC1-5 on the substrate. Together these findings suggest that the regulation of C-cadherin by activin and its activation by mAb AA5 involve changes in its cellular organization or interactions with other cell components that are not intrinsic to the isolated protein.

Show MeSH
Related in: MedlinePlus